The present invention pertains to methods, reagents, compositions, kits, and apparatus for use in ligating substantially contiguous ligands together on a target template. In particular, the present invention relates to methods, reagents, compositions, and kits for performing deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) hybridization assays.
The following definitions are provided to facilitate an understanding of the present invention.
The term "target" or "target molecule" refers to a molecule of interest, i.e. the molecule whose presence one wishes to know. The target is a member of a biological binding pair.
The term "biological binding pair" as used in the present application refers to any pair of molecules which exhibit mutual affinity or binding capacity. For the purposes of the present application, the term "ligand" will refer to one molecule of the biological binding pair, and the term "antiligand" or "receptor" will refer to the opposite molecule of the biological binding pair. For example, without limitation, embodiments of the present invention have application in nucleic acid hybridization assays where the biological binding pair includes two complementary strands of polynucleic acid. One of the strands is designated the ligand and the other strand is designated the antiligand or receptor. One of the strands may also be a target molecule. The designation of ligand or antiligand is a matter of arbitrary convenience. The biological binding pair may include antigens and antibodies, drugs and drug receptor sites, and enzymes and enzyme substrates, to name a few. A biological binding pair is capable of forming a complex under bindings conditions.
The term "probe" refers to a ligand of known qualities capable of selectively binding to a target antiligand or receptor. As applied to nucleic acids, the term "probe" refers to a strand of nucleic acid having a base sequence complementary to a target strand. The probe and the target are capable of forming a probe target complex under binding conditions.
The term "label" refers to a molecular moiety capable of detection including, by way of example, without limitation, radioactive isotopes, enzymes, luminescent agents, precipitating agents, and dyes. The term "agent" is used in a broad sense, including any molecular moiety which participates in reactions which lead to a detectable response. The term "cofactor" is used broadly to include any molecular moiety which participates in reactions with the label.
The term "amplify" is used in the broad sense to mean creating an amplification product, which may include by way of example, additional target molecules, or target-like molecules, capable of functioning in a manner like the target molecule, or a molecule subject to detection steps in place of the target molecule, which molecules are created by virtue of the presence of the target molecule in the sample. In the situation where the target is a polynucleotide, additional target, or target-like molecules, or molecules subject to detection can be made enzymatically with DNA or RNA polymerases.
The term "reactive functional group" refers to a functional group capable of forming a covalent bond upon activation between two ligands held in a reactive position. Activation may include chemical or physical changes to the environment.
The term "photoreactive functional group" refers to a reactive functional group capable of forming a covalent bond upon photoactivation with radiant energy between two ligands held in a reactive position.
An example of a photoreactive functional group includes, without limitation, olefins, conjugated olefins, ketones, .alpha., .beta.-unsaturated ketones, azides, conjugated polyolefins characterized by conjugated double bonds and ketone functionality and aromatic compounds. Photoreactive functional groups can be further characterized as coumarins, psoralens, anthracenes, pyrenes, carotenes, tropones, chromones, quinones, maleic anhydride, alkyl maleimide and derivatives thereof. Further examples of photoreactive functional groups can be found in the reference J. G. Calvert, James N. Pitts, Jr., Photochemistry, pages 536-48, John Wiley Sons, Inc. (1966), incorporated by reference herein.
The term "contiguous" means an adjacent area of a molecule. By way of example, in the case of biological binding pairs, where a first ligand binds to a receptor target molecule, the area surrounding and adjacent to the first ligand is open and capable of binding to a second ligand contiguous to the first. In the context of polynucleotides, where a first probe binds to an area of a polynucleotide target molecule, an adjacent mutually exclusive area along the length of the target molecule can bind to a second probe which will then be contiguous to the first. The target molecule acts as a template, directing the position of the first probe and the second probe. The term "substantially contiguous" is used in the functional sense to include spatial orientations which may not touch, may not abut, or may overlap yet function to bring a reactive covalent functional group into a reactive position.
The term "capture ligand" means a ligand capable of specifically binding with a capture antiligand associated with a support.
The term "retrievable support" is used in a broad sense to describe an entity which can be substantially dispersed within a medium and removed or separated from the medium by immobilization, filtering, partitioning, or the like.
The term "support", when used alone, includes conventional supports such as filters and membranes as well as retrievable supports.
The term "reversible", in regard to the binding of ligands and antiligands, means capable of binding or releasing upon imposing changes which do not permanently alter the gross chemical nature of the ligand and antiligand. For example, without limitation, reversible binding would include such binding and release controlled by changes in pH, temperature, and ionic strength which do not destroy the ligand or antiligand.
Genetic information is stored in living cells in thread-like molecules of DNA. In vivo, the DNA molecule is a double helix, each strand of which is a chain of nucleotides. Each nucleotide is characterized by one of four bases: adenine (A), guanine (G), thymine (T), and cytosine (C). The bases are complementary in the sense that, due to the orientation of functional groups, certain base pairs attract and bond to each other through hydrogen bonding and .pi.-stacking interactions. Adenine in one strand of DNA pairs with thymine in an opposing complementary strand. Guanine in one strand of DNA pairs with cytosine in an opposing complementary strand. In RNA, the thymine base is replaced by uracil (U) which pairs with adenine in an opposing complementary strand.
The genetic code of a living organism is carried upon the DNA strand in the sequence of base pairs. DNA consists of covalently linked chains of deoxyribonucleotides and RNA consists of covalently linked chains of ribonucleotides.
Each nucleic acid is linked by a phosphodiester bridge between the 5'-hydroxyl group of the sugar of one nucleotide and the 3'-hydroxyl group of the sugar of an adjacent nucleotide. Each linear strand of naturally occurring DNA or RNA has one terminal end having a free 5'-hydroxyl group and another terminal end having a 3'-hydroxyl- group. The terminal ends of polynucleotides are often referred to as being 5'-termini or 3'-termini in reference to the respective free hydroxyl group. Naturally occurring polynucleotides may have a phosphate group at ,the 5'-terminus. Complementary strands of DNA and RNA form antiparallel complexes in which the 3'-terminal end of one strand is oriented and bound to the 5'-terminal end of the opposing strand.
Nucleic acid hybridization assays are based on the tendency of two nucleic acid strands to pair at their complementary regions. Presently, nucleic acid hybridization assays are primarily used to detect and identify unique DNA or RNA base sequences or specific genes in a complete DNA molecule, in mixtures of nucleic acid, or in mixtures of nucleic acid fragments.
The identification of unique DNA or RNA sequences or specific genes within the total DNA or RNA extracted from tissue or culture samples, may indicate the presence of physiological or pathological conditions. In particular, the identification of unique DNA or RNA sequences or specific genes, within the total DNA or RNA extracted from human or animal tissue, may indicate the presence of genetic diseases or conditions such as sickle cell anemia, tissue compatibility, cancer and precancerous states, or bacterial or viral infections. The identification of unique DNA or RNA sequences or specific genes within the total DNA or RNA extracted from bacterial cultures may indicate the presence of antibiotic resistance, toxicants, viral- or plasmid-born conditions, or provide identification between types of bacteria.
Thus, nucleic acid hybridization assays have great potential in the diagnosis and detection of disease. Further potential exists in agriculture and food processing where nucleic acid hybridization assays may be used to detect plant pathogenesis or toxicant-producing bacteria.
One of the most widely used polynucleotide hybridization assay procedures is known as the Southern blot filter hybridization method or simply, the Southern procedure (Southern, E., J. Mol. Biol., 98, 503, 1975). The Southern procedure is used to identify target DNA or RNA sequences. The procedure is generally carried out by subjecting sample RNA or DNA isolated from an organism, potentially carrying the target sequence of interest, to restriction endonuclease digestion to form DNA fragments. The sample DNA fragments are then electrophoresed on a gel such as agarose or polyacrylamide to sort the sample fragments by length. Each group of fragments can be tested for the presence of the target sequence. The DNA is denatured inside the gel to enable transfer to nitrocellulose sheets. The gel containing the sample DNA fragments is placed in contact (blotted) with nitrocellulose filter sheets or diazotized paper to which the DNA fragments transfer and become bound or immobilized. The nitrocellulose sheet containing the sample DNA fragments is then heated to approximately 85.degree. C. to immobilize the DNA. The nitrocellulose sheet is then treated with a solution containing a denatured (single-stranded) radio-labeled DNA probe. The radio-labeled probe includes a strand of DNA having a base sequence complementary to the target sequence and having a radioactive moiety which can be detected.
Hybridization between the probe and sample DNA fragments is allowed to take place. During the hybridization process, the immobilized sample DNA is allowed to recombine with the labeled DNA probe and again form double-stranded structures.
The hybridization process is very specific. The labeled probe will not combine with sample DNA if the two DNA entities do not share substantial complementary base pair organization. Hybridization can take from 3 to 48 hours, depending on given conditions.
Unhybridized DNA probe is subsequently washed away. The nitrocellulose sheet is then placed on a sheet of X-ray film and allowed to expose. The X-ray film is developed with the exposed areas of the film identifying DNA fragments which have hybridized to the DNA probe and therefore have the base pair sequence of interest.
The use of nucleic acid hybridization assays has been hampered in part due to the inability of investigators to detect a signal from a probe indicating the presence of target over background caused by nonspecific binding of the probe to non-target entities. The signal to noise ratio can be improved by decreasing background noise by segregating the nucleic acid from other cellular debris. Background noise is sometimes reduced by subjecting a sample, potentially containing the nucleic acid of interest, to which probe has been added, to stringent washes which will remove probe which is only nonspecifically bound. However, as a practical matter, a small amount of probe is disassociated from the target with each wash, particularly as stringency increases. Investigators must balance between limiting background noise and retaining enough signal to detect target.
The signal to noise ratio may also be improved by increasing or amplifying the signal which can be generated from a single target molecule. As a practical matter, background noise is usually amplified to some degree with the signal. Investigators must balance between amplifying signal and maintaining background noise to a level at which the target can be detected.
The techniques used to improve signal-to-noise ratios in nucleic acid assay systems must be compatible with the overall environment in which the system is to be used. For research purposes, time may be of less importance than maximizing a signal-to-noise ratio. However, in medical applications and in food testing, time may be a critical factor. For example, time considerations, in part, are driving immuno and nucleic acid diagnostics away from radioactive labels to nonisotopic systems. Nonisotopic systems do not require long development times necessary for X-ray film. Time considerations are also driving medical and food diagnostics away from culture techniques to other systems of detection.
The problem of developing a useful and reliable signal-to-noise ratio has led researchers to several techniques. Yabusaki et al. in PCT Patent Application PCT/US84/02024, International Publication Number WO85/02628 entitled Nucleic Acid Hybridization Assay report the use of photoreactive crosslinking reactions to link a probe to target DNA. However, the Yabusaki reference does not suggest means for amplifying the signal or photoreactively forming covalent bonds between contiguous ligands on a target template. European Patent Application 85308910.0 to Hunkapiller et al. entitled Detection of Specific Sequences in Nucleic Acids suggests that two contiguous probes can be ligated together enzymatically to form a detectable unit. However, enzyme ligations are inefficient, particularly, at low target concentrations normally encountered in analytical applications, and enzymatic ligation is time-consuming. Enzymatic ligation requires many complex steps and is subject to variations in enzyme activity. Enzymes require two hours or more for proper incubation. Two hours of incubation added to other assay steps, including sample preparation, hybridization, and detection, enlarges the total duration of the assay system limiting its use in many clinical settings.